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The online version of this article (doi:10.​1186/​1476-4598-11-84) contains supplementary material, which is available to authorized users.

Competing interests

The authors report no conflict of interest.

Authors' contributions

NG-K participated in the design of the study and performed most of the experiments. IM was responsible for obtaining the ascites, performing the immunohistochemistry staining with the TMA and obtaining the clinical data. DL performed the long term cell viability assays. CR participated in the design of the study and helped to draft the manuscript. AP conceived the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Ascites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.

Methods

Using human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.

Results

In the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.

Conclusions

These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

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Zusatzmaterial

Additional file 1: Figure S1. - (A) Time course of Mcl-1 protein expression in OVCAR3 cells following addition of 10% OVC509 and OVC551 ascites to the cell culture media. Cells were incubated with ascites for 2 h and 4 h and proteins were extracted. Immunoblots were probed with anti-Mcl-1 and anti-tubulin antibodies (as a loading control). (B) Real-time PCR analysis of Mcl-1 transcript levels from CaOV3 cells incubated in the presence or absence of OVC415 and OVC509 ascites (10%) and actinomycin D. Results were standardized using primers of the housekeeping gene RPLPO. Results are expressed as fold change relative to basal levels observed in cells incubated in the absence of ascites. (C) OVCAR3 cells were treated with protein synthesis inhibitor cycloheximide 2 h before addition of OVC509 ascites. Lysates were obtained after 3 h and immunoblot analysis of Mcl-1 were performed. Figure S2. – (A) CaOV3 cells were transfected with vehicle, Mcl-1 or control siRNA. Mcl-1 knockdown was assessed by immunoblot analysis 24 h and 48 h after transfection. ERK1/2 was used as a loading control. (B) CaOV3 were transfected as described and Bcl-2 and Bcl-XL expression were determined 24 h after transfection of Mcl-1 siRNA to ensure that Mcl-1 knockdown does not altered Bcl-2 and Bcl-XL expression. Figure S3. – (A) CaOV3 cells were incubated with various ascites (10%) for 2 h. Akt phosphorylation and expression were then determined by immunoblot. (B) CaOV3 cells were incubated with OVC 439 ascites and Akt phosphorylation and expression were assessed at 2 h and 4 h after addition of ascites. (PPTX 245 KB)